15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and cumene-hydroperoxide

At functional luteolysis, the rat corpus luteum generates hydrogen peroxide (H2O2), which is known to rapidly inhibit gonadotropin-sensitive cAMP and progesterone production in isolated luteal cells. Lipid peroxides also increase markedly in the rat corpus luteum with the onset of functional luteolysis, and H2O2 is a potent inducer of lipid peroxidation. However, the actions of lipid peroxides on cell function are unknown. The objective of this study was to investigate the impact of typical lipid peroxides, cumene hydroperoxide (CuOOH) and 15(S)-hydroperoxyeicosatetraenoic acid, on rat luteal cells. CuOOH inhibited both LH-sensitive cAMP accumulation (ED50, 25 microM) and progesterone production (ED50, 20 microM). 15(S)-hydroperoxyeicosatetraenoic acid also dose dependently inhibited steroidogenesis. A significant reduction of LH-stimulated progesterone production was evident within 5 min of treatment with CuOOH, whereas inhibition of cAMP accumulation was not evident until 60 min. 8-Bromo-cAMP and 22-hydroxycholesterol caused partial and complete reversal of CuOOH-inhibited progesterone secretion, respectively. Preincubation of cells with o-phenanthroline completely reversed the inhibitory effects of CuOOH on cAMP accumulation and partially reversed its effects on progesterone production. Incorporation of radiolabeled amino acids into luteal proteins was significantly inhibited by CuOOH (25 microM) within 2 min of treatment and was reduced to 40 +/- 14% of control levels at 60 min. CuOOH (25 microM) maximally stimulated PGE2 production within 30 min of treatment (180 +/- 30% of control), a response that was completely blocked by aristolochic acid (100 microM), a phospholipase-A2 inhibitor, and indomethacin (1 microgram/ml), a prostaglandin (PG) synthesis inhibitor. The present results suggest that the acute inhibitory action of lipid peroxides on LH-stimulated progesterone production occurs down-stream of cAMP synthesis and appears to be due to impaired cholesterol utilization for steroidogenesis, possibly through inhibition of protein synthesis. The stimulation of PGE2 production by CuOOH appears to involve the activation of phospholipase-A2, which is a rate-limiting step in PG synthesis. Lipid peroxides as well as H2O2 may serve as mediators of functional luteolysis.

Topics: Animals; Benzene Derivatives; Corpus Luteum; Cyclic AMP; Dose-Response Relationship, Drug; Female; Gonadotropins; Leukotrienes; Lipid Peroxides; Luteinizing Hormone; Phenanthrolines; Progesterone; Rats; Rats, Inbred Strains; Steroids

Rat basophilic leukemia cells have frequently been employed for investigating the pathways of leukotriene biosynthesis, a class of biologically active arachidonic acid metabolites. However, information is lacking on the levels of selenium-dependent glutathione peroxidase (Se-GSH-Px), non-Se-GSH-Px and glutathione S-transferases (GSH-S-Trs), key enzymes involved in fatty acid hydroperoxide metabolism and leukotriene biosynthesis in these cells. Both GSH-S-Trs and non-Se-GSH-Px reactions are catalyzed by the same enzyme. In the present studies, we have measured the enzyme activities of GSH-Px(s) and GSH-S-Trs in the 105,000 X g supernatant fraction of sonified RBL-1 cells. The specific activities for GSH-Px(s) toward H2O2, cumene hydroperoxide, and 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) are 12.6, 17.9 and 26.9 nmoles X min-1 X mg-1 protein, respectively. A specific activity of 18.9 nmoles X min-1 X mg-1 protein with 1-chloro-2,4-dinitrobenzene was estimated for the GSH-S-Trs. Therefore, the cell fraction that exhibits 5-lipoxygenase activity also contains selenium and non-selenium glutathione peroxidases.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Basophils; Benzene Derivatives; Cells, Cultured; Glutathione; Glutathione Peroxidase; Glutathione Transferase; Hydrogen Peroxide; Leukemia, Experimental; Leukotrienes; Lipid Peroxides; Rats; Selenium